Actin Filament Structure

Contraction of muscle occurs when myosin heads pull on the thin (actin-containing) filaments, causing them to slide past the myosin filaments. The thin filaments are involved not only in contraction, but also in its regulation via the actin-binding proteins, tropomyosin and troponin. We are studying the structural basis of the regulatory switching of thin filament activity.

Steric blocking of myosin head binding by tropomyosin switches off thin filaments

In collaboration with Dr. W. Lehman (Boston University School of Medicine) we have used negative staining of thin filaments (left image) and helical reconstruction techniques to study thin filament structure. Our reconstructions demonstrate that the elongated tropomyosin molecule, which runs along the actin helix, physically blocks the binding of myosin heads to the thin filament in the off-state by covering the myosin binding site (middle and right images). When muscle is activated, Ca2+ binds to troponin, causing tropomyosin to move. This partially uncovers the myosin binding site, allowing heads to bind, thus initiating contraction. When myosin binds, it pushes tropomyosin further, fully opening the myosin binding site and allowing cooperative switching on of the thin filament. These results directly confirm the “steric-blocking” model of regulation, which had previously been postulated based on X-ray diffraction data from muscle. In numerous subsequent studies, we have continued our collaboration with Dr. Lehman and others and used mutant tropomyosin, troponin and actin to elucidate the basic mechanism of this regulatory movement and the ways in which disease-related mutations in these proteins would affect this regulatory mechanism. Current studies are directed at obtaining an atomic model of the thin filament including actin, tropomyosin and troponin.

Actin filaments edATmedATm oblique

Negatively stained thin filaments (left) together with 3D reconstruction in longitudinal and oblique views showing actin subunits (gold) and tropomyosin in three different positions: red, off-state (low Ca2+); yellow, Ca2+ -induced state; green, myosin-induced state.